ENZYME COFACTORS

Ms M. Mombeshora
Biochem Lecture 3
 Many Enzymes Require
Cofactors for Activity
• A cofactor is a small non-protein molecules
that is bound (either tightly or loosely) to an
enzyme and is required for catalysis

• Catalytic activity of many enzymes depends

on the presence of cofactors
Many Enzymes Require
Cofactors for Activity
 Cofactors
• Some cofactors can be made inside the body,
such as ATP
• Others must be consumed in food
• Minerals, for example, come from the
environment
• Minerals cannot be made from scratch by any
living cell
 Cofactors
• The organic compounds we refer to as
“vitamins” are cofactors that our own bodies
cannot make
• Vitamins must be consumed from food
• Vitamins assist our cells to be able to perform
essential life functions
 Cofactors
At biochemical level

• Cofactors are important in understanding how

biological reactions proceed

• The presence or absence of cofactors may

determine how quickly reactions proceed
from their reactant to their product
 Cofactors
At biological level

• Understanding cofactors is important to

understanding health

• Without the proper cofactors, humans and

other animals can develop serious diseases
and even death
 Function of Cofactors

• Generally serve the purpose of supplying:

i. chemical groups
or
ii. properties that are not found in other chemical
groups
• ATP, for example, is a cofactor with a unique
ability to transfer energy to drive chemical
processes
• Chemical processes like the activity of enzymes
and transport proteins
 Function of Cofactors

Heme

• Is a chemical complex that contains iron

• Iron allows heme to bond to oxygen molecules
in a unique way
• Heme is necessary for our blood cells to carry
oxygen through our bodies
 Function of Cofactors

• There are dozens of known cofactors, each of

which may be necessary for multiple biochemical
reactions
• As a result, the functions of cofactors may be as
diverse as their chemical structures and
properties
• The wide-ranging effects of cofactors can be seen
by studying vitamin deficiencies
• Deficiencies of different vitamins, many of which
are cofactors, have many different negative
effects on human health
 Types of Cofactor

1. Vitamins
• Are organic compounds that are co-factors for
necessary biochemical reactions
• Typically need to be consumed in the diet,
because they cannot be made inside the body
• Many are cofactors which help enzymes to
catalyse reactions, such as the production of
important proteins
• Vitamin C, for example, is a cofactor for the
production of the connective tissue collagen
Types of Cofactor

1. Vitamins
• This is why people who get scurvy – a severe
form of vitamin C deficiency – may experience
connective tissue problems, including:
– muscle weakness
– muscle soreness
– even unexplained bleeding as the connective tissues
of blood vessels cannot be replaced
 Types of Cofactor

1. Vitamins
• Vitamin deficiencies are a good illustration of
the effects of co-factor deficiency
• Just as there are many possible vitamin
deficiencies with many different symptoms
• There are many different co-factors that our
body needs to carry out its diverse necessary
biochemical reactions
 Types of Cofactor
1. Vitamins
• The body’s requirement for diverse
vitamin cofactors is also the reason
why nutritionists counsel people to
“eat the rainbow”

• Many plants’ colours are produced

by cofactors

• So by eating fruits and vegetables

in a wide variety of colours helps to
ensure that we consume a healthy
variety of cofactors
 Types of Cofactor
2. Minerals
• Like vitamins, minerals are chemicals from
outside of the body
• They must be ingested to allow our cells to
function properly
• The difference is that while vitamins are organic
molecules – molecules containing carbon, which
are often made by other living things
• Minerals are inorganic substances that occur
naturally, and are often found in rocks and soil
 Types of Cofactor
2. Minerals

• Minerals often enter our diets from plants

• Plants draw them up out of the ground through
their roots along with water
• In some rare cases, people with vitamin
deficiencies may feel the urge to eat certain types
of soil to obtain the minerals from the soil directly
 Types of Cofactor
2. Minerals
• Minerals that are important for human health
include:
i. copper, which is necessary for the function of some
important liver enzymes that break down toxins
ii. iron, which is necessary for the function of some
important metabolic enzymes
iii. magnesium, which is necessary for the function of
DNA polymerase and other enzymes
iv. zinc, which is also necessary for DNA polymerase as
well as some liver enzymes
 Types of Cofactor
2. Minerals
• As with vitamins, there can be too much of a good
thing
• While minerals are necessary in small amounts for our
metabolisms to function, taking large doses of them
can result in toxicity and death
• Indeed, overdoses of iron-containing multivitamins are
a leading cause of death in children under 4
• Children may mistake these multivitamins for candy
 Types of Cofactor
3. Organic Non-Vitamin Cofactors
• Some cofactors are organic substances not
classified as enzymes
• Some of these may be made inside our own
bodies, and so not qualified as vitamins
 Types of Cofactor
3. Organic Non-Vitamin Cofactors
• Organic non-vitamin cofactors include ATP – an
essential assistant to many biochemical processes
• ATP transfers energy to numerous enzymes,
transport proteins, and more
• Coenzyme Q, which plays a vital role in the
mitochondrial transport chain
• Heme, which is a complex iron-containing
compound that is necessary for our blood cells to
carry oxygen throughout our bodies
 Examples of cofactors
Thiamine (Vitamin B3)
• A vitamin found primarily in edible seeds such as
beans, corn, and rice
• To improve public health, thiamine is often
artificially added to wheat-containing products
such as breakfast cereals
• In the body, thiamine is used to make many co-
enzymes that assist with important processes
• It is made into thiamine pyrophosphate, which is
necessary to break down sugars and amino acids
 Examples of cofactors
Thiamine (Vitamin B1)
• Severe thiamine deficiency is one cause of Korsakoff
Syndrome – a rare neurological disorder seen in people
with severe alcohol addiction
• In Korsakoff Syndrome, severe malnutrition, lack of
thiamine, and brain damage from overuse of alcohol
combine to produce severe symptoms, including
memory impairment
• Some sufferers of Korsakoff Syndrome are not able to
form new memories because the metabolism of their
brain is so impaired
 Examples of cofactors
Folic Acid (Vitamin B9)
• Folic acid is another vitamin which is
now often added to food to improve public
health
• It is necessary for the body to produce DNA,
RNA, and amino acids, which are necessary for
growth and cell division.
• This makes folic acid particularly essential for
pregnant women, whose foetuses are
producing new cells and tissues very quickly
 Examples of cofactors
Folic Acid (Vitamin B9)
• Deficiencies in folic acid can lead to:
– birth defects in babies, or
– to anaemia in pregnant women who may not be able to
make enough new blood cells to supply both them and the
baby
• For this reason, it is recommended that all women of
childbearing age talk to their doctors about taking folic acid
supplements
• Pregnancy outcomes are best when sufficient folic acid is
present in the mother’s body even before pregnancy begins
 Co-enzyme derived from Vitamins of B
complex
vitamins active form (co-enzyme)

Thiamine Vitamin B 1 TPP (thiamine pyrophosphate)

Riboflavin Vitamin B 2 FMN, FAD
Niacin Vitamin B 3 NAD,NADH
Pantothenic acid Vitamin component of coenzyme
B5 A
Pyridoxine Vitamin B 6 PLP (pyridoxal phosphate)
Biotin Biotin
Folic acid THF (Tetrahydrofolate)
Cobalamine Vitamin B 12 cobamide
 Deficiencies and Sources
Vitamins
preformed: liver, egg yolk, butter, milk
A - night blindness b-carotene: dark green and yellow veggies
B1 - beri-beri seeds, nuts, wheatgerms, legumes, lean meat
B2 - pellagra meats, nuts, legumes
B3 - pellagra meats, nuts, legumes
B5 - none known yeast, grains, egg yolk, liver
yeast, liver, wheatgerm, nuts, beans, bananas
B6 - neurologic disease
corn, soy, egg yolk, liver, kidney, tomatoes
B7 - widespread injury
B9 - anemia yeast, liver, leafy veggies

B12 - pernicious anemia liver, kidney, egg, cheese

C - scurvy citrus and soft fruits

milk, fortified food, fish oils, egg
D - ricketts, osteomalacia yolks, liver
E - neurologic?, hemolytic anemia veggie oils, nuts
green leafy veggies, fruits, dairy products, veggie oils,
K - bleeding disorders cereals, meats
ENZYME KINETICS
 Enzyme Assay
• The act of measuring how fast a given (or
unknown) amount of enzyme will convert
substrate to product
• The act of measuring a velocity
• How fast a given amount of substrate is
converted to a product depends on [enzyme]
present
• By measuring [product] formed in a given
time, the [enzyme] can be determined
 Enzyme Assay
• Requires a way to determine [product] or
[substrate] at a given time after starting the
reaction
• If the product and substrate have different UV
or visible spectra, fluorescence spectra, e.t.c.
• The progress of the reaction can be followed
by measuring the change in the spectrum with
time
 Enzyme Assay
• If there are no convenient spectral changes,
physical separation of substrate and product
may be used
• Radioactivity may also be utilised
 Velocity (v)
• The change in substrate or product concentration
per time
• Also termed:
 Rate
 The first derivative of the [product] or [substrate]
with respect to time, d[P]/dt
• Enzyme velocity is measured in molar per minute
(M/min) or more usually in micromolar per
minute (µM/min).
Velocity (v)
 Initial Velocity (v0)
• Measurement of the rate under conditions
where there is no significant change in the
[substrate]
• The velocity is not necessarily the same at all
times after you start the reaction
• The depletion of substrate, inhibition by the
product, or instability of the enzyme can cause
the velocity to change with time
Initial Velocity (v0)
 Reaction mechanism

Approaching zero order

First order
 Michaelis-Menten equation
• Hyperbolic kinetics, saturation kinetics

Vo = Vmax [S]
Km + [S]
• Velocity depends on substrate concentration
when [S] is low but
• Does not depend on substrate concentration
when [S] is high.
Michaelis-Menten equation
 Michaelis-Menten equation
• Substrate concentration affects the velocity of
an enzyme catalysed reaction
• Almost all enzyme-catalysed reactions show
saturation behaviour
• At a high enough substrate concentration, the
reaction just won’t go any faster than Vmax
• The substrate concentration required to
produce a velocity that is one-half of Vmax is
called the Km.
 Michaelis-Menten Graph

Approaching
Zero order

First order

Plot of substrate concentration versus reaction velocity

 Michaelis-Menten

Maud Mentein Leonor Michaelis

 Enzyme Kinetics - the study of the
rates of reactions
Enzymes
• Endow cells with the remarkable capacity to exert kinetic
control over thermodynamic potential
• The agents of metabolic function

What we want to be able to determine:

• Maximum velocity
• Substrate affinity
• Inhibitor affinity

What can we do with the information?

• Control and manipulate metabolic events
 The Michaelis-Menten Equation

• Allows one to easily calculate enzyme reaction

velocities without having to refer to the graph
every time

• Two constants (specific for each enzyme)

Vmax and Km, the Michaelis-Menten constant
 The Michaelis-Menten Equation

• Vmax – the maximum velocity

 Velocity where enzyme is saturated with substrate

• Km -substrate concentration at half the maximal

velocity i.e. when [S] = Km, Vo yields Vmax/2 = Km.
 A measure of the affinity of the enzyme for its
substrate
 Low Km value = High affinity, High Km value = Low
affinity
 The Michaelis-Menten Equation

• Km -affinity of the enzyme for the substrate,

• Kcat -catalytic ability of the enzyme.

• Kcat/Km, is the specificity constant -a measure of

how good the enzyme is at its job.

• A high specificity constant means that the reaction

goes fast (Kcat is big) and does not need a high
concentration of substrate (Km is small).
 Enzyme assays
Turnover number Kcat
• The maximum number of moles of substrate that an
enzyme can convert to product per catalytic site per unit
time
At saturation point [S], maximum rate
Vmax = Kcat[Et]
• Where Kcat is the rate constant for ES P in these
conditions (only process occuring since enzyme is saturated
• [Et] is the total enzyme concentration (i.e no. of active sites
The double reciprocal plot
(Lineweaver-Burk)

1  KM  1 1
   
vo  Vmax  S Vmax
 Lineweaver-Burk plot: slope = KM/Vmax,
1/vo intercept is equal to 1/Vmax
the extrapolated x intercept is equal to -1/KM
For small errors in at low [S] leads to large errors in 1/vo

kcat is how many reactions an

enzyme can catalyze per second
Vmax
kcat 
ET The turnover number
Competitive Inhibition: Lineweaver-Burke Plot
Uncompetitive Inhibition
Uncompetitive Inhibition: Lineweaver-Burke Plot
Mixed inhibition